In bacteria, the 450 kDa RNA polymerase (RNAP) holoenzyme, comprising the evolutionarily conserved catalytic core combined with the initiation-specific subunit, directs transcription initiation. Bacterial transcription depends on a primary factor that is essential for viability. In addition, most bacteria contain multiple alternative 'sthat control regulons in response to environmental cues. The principal control point of gene expression in bacteria is transcription initiation, and a major mechanism by which bacteria regulate transcription initiation is through regulation of activity. Dramatic insights have come from structural studies of bacterial RNAPs. Nevertheless, many challenges remain, including understanding the complex process of transcription initiation and its control by factors and modulators of -factor function. Specifically, we propose to: 1. Determine the structural basis for the recognition of the -10 element by alternative 's. In the last project period, we determined high-resolution crystal structures of a primary bound to the - 10 element sequence. How do alternative 's recognize and facilitate melting of their cognate -10 elements, which bear no relationship to the primary -10 element? To address this question, we will determine structures of alternative 's bound to their -10 element sequence. 2. Structurally characterize the role of N region I in promoter recognition. The N family of factors (also called 54) is evolutionarily unrelated to the 70 family and functions through a distinct mechanism. In the last funding period, we determined crystal structures of N/promoter DNA complexes, but these structures lack the N-terminal N region I, which controls promoter melting. We will determine crystal structures of full-length N bound to promoter DNA constructs. 3. Determine the structure of a Sau phage G1 ORF67/Sau A4/DNA complex. In the last funding period, our studies revealed that ORF67 targets transcription in a promoter-specific manner by disrupting RNAP -subunit/UP-element interactions, but the molecular basis for how this is achieved was not clear from our ORF67/A4 structures. To understand the molecular details of ORF67 function, we will determine the crystal structure of an ORF67/A4/promoter DNA ternary complex. 4. Determine the structure of a new family of anti-: Streptomyces venezuelae BldN/RsbN. An ECF /anti- pair, BldN/RsbN, plays a critical role in the cellular differentiation of Streptomyces into aerial hyphae and spores. The biological cue sensed by RsbN to release BldN is unknown, and sequence analysis also indicates RsbN represent a new class of anti-. A crystal structure of the BldN/RsbN complex will help address these questions.